Hemorrhagic shock is the clinical condition of inadequate perfusion (ischemia) of the tissues as a result of blood loss. The treatment of hemorrhagic shock requires prompt restoration of tissue perfusion by infusing large volumes of lactated Ringer's solution, saline, albumin solutions, whole blood, dextran, or solutions containing stabilized hemoglobin. Despite prompt restoration of intravascular volume, the morbidity and mortality due to hemorrhagic shock remain unacceptably high.
A key reason for the frequent failure of fluid resuscitation in hemorrhagic shock is the generalized and progressive microvascular injury which is a consequence of tissue ischemia. The microvasculature consists of venules, arterioles and their intervening capillary bed. Following a period of ischemia and reperfusion (as occurs in hemorrhagic shock followed by fluid resuscitation), the capillaries and venules become excessively permeable and the arterioles constrict. The leaking of blood proteins from the excessively permeable venules into the tissue interstitium causes additional fluid loss and edema (swelling), which further compromises blood flow and leads to organ dysfunction. Arteriolar constriction results in areas of persistent tissue ischemia and local hypoxia. Because of a forced state of anaerobic metabolism resulting in lactic acid accumulation and tissue acidosis, hemorrhagic shock may be followed by multiple organ failure and death several days after prompt and seemingly successful restoration of circulating volume sufficient to reperfuse vital organs.
In addition to the arteriolar constriction and the increased permeability of venules, a third factor in microvascular dysfunction is the adhesion of circulating white blood cells (leukocytes) to venular endothelial cells. Adherent leukocytes release proteolytic enzymes, inflammatory mediators and reactive oxidants, all of which further accelerate the cascade of microvascular injury. Leukocytes, specifically polymorphonuclear neutrophils and monocytes, originate in bone marrow. From this location they enter the general circulation and adhere to post-capillary venules at sites of the inflammatory response. They initially adhere to the endothelial surface of these venules and eventually migrate across the vessel wall into the tissues. The process of leukocyte recruitment into an area of inflammatory response begins with margination of leukocytes to the slower peripheral section of the bloodstream. At this stage the leukocytes loosely adhere to the endothelium, and this weak binding results in "rolling" of the leukocytes along the vessel wall. In this sense, the leukocytes sample the local environment and are fully arrested if the appropriate signals are received, resulting in stronger adhesive interactions. Following firm adhesion, the leukocytes transmigrate across the endothelial wall. They then continue through the tissues to sites of inflammation where they play a key role in killing of bacteria. Leukocyte adhesion is an early sign of microvascular injury which occurs during the body's response to injury or infection. When the microvascular injury is localized to a small area, there are usually no adverse effects to the person. However, when generalized microvascular injury occurs throughout the body, vascular permeability may be impaired to such an extent that organ injury and death result.
At a molecular level, an important cause of microvascular injury following hemorrhagic shock is the generation of highly reactive oxidants. These toxic species are formed in ischemic tissues following the reintroduction of oxygen when perfusion has been restored by fluid resuscitation. One critical location of oxidant generation responsible for microvascular injury is the endothelial cell, a single layer of which forms the inner lining of all blood vessels. Endothelial cells actively regulate microvascular function through the production of several vasoactive substances, including nitric oxide (NO).
Nitric oxide's normal role is to regulate the degree of constriction of the microvessels, their permeability and to inhibit the adhesion of circulating leukocytes. When NO levels decrease below normal, arterioles constrict, venules become permeable, and leukocytes adhere to venular walls. Reactive oxidants, such as the superoxide radical, may trigger microvascular dysfunction by reacting with NO to form peroxynitrite, which is even more toxic than the superoxide radical. The net decrease in microvascular NO resulting from this reaction may be a key cause of the arteriolar constriction, as well as the increased venular permeability and leukocyte adhesion seen in the setting of ischemia/reperfusion.
Because oxidant species play a central role in microvascular dysfunction, investigators have experimented with antioxidants as a way of mitigating the effects of reactive oxidants. Superoxide dismutase (SOD), catalase, and the iron binding molecule deferoxamine have been tried in resuscitation fluids after hemorrhagic shock with limited success. Adverse side effects or poor lipid solubility have been proposed to explain why some agents capable of destroying reactive oxidants in vitro have proven less effective in a living organism. High lipid solubility is a crucial characteristic of a clinically effective antioxidant because oxidant generation takes place within the cells, for example, within mitochondria which are intracellular organelles inside of the endothelial cells. A rapid, passive diffusion of antioxidants is possible only if the antioxidant can pass through the lipid membranes investing the endothelial cells and their mitochondria.
The ideal antioxidant for mitigating ischemia/reperfusion microvascular injury would be one which is lipid soluble and non-toxic. .alpha.-lipoic acid is a lipid soluble antioxidant which naturally occurs in the body. Even in the pharmacological doses given to patients with diabetic peripheral nerve disease, .alpha.-lipoic acid has been administered safely and without long term side effects.
Alpha-lipoic acid, in addition to its non-toxicity and lipophilicity, has the advantage of being rapidly converted in tissues into its reduced form, dihydrolipoic acid (DHLA). DHLA also has potent antioxidant effects. Further, both .alpha.-lipoic acid and DHLA can disarm oxidants through a variety of mechanisms including free radical quenching, metal chelation, and regeneration of other common natural antioxidants.
Because resuscitation fluids--saline, dextran, blood, stabilized hemoglobin solutions--are all aqueous solutions, a problem with lipid soluble antioxidants, such as .alpha.-lipoic acid is its poor water solubility. The solubility may be enhanced by adding benzyl alcohol or DMSO, but such solvents introduce additional side effects and potential toxicities. The present invention overcomes the problem of delivering a lipophilic antioxidant in an aqueous resuscitation fluid by a method of ultrasonic solubilization which forms a stable micellular solution of .alpha.-lipoic acid. The utility of the inventive method described herein will be illustrated in protocols showing the effect of ultrasonicated aqueous solutions of .alpha.-lipoic acid and other antioxidants on leukocyte adhesion, venular permeability and arteriolar constriction, following ischemia/reperfusion.
Previous methods of delivering antioxidants which are lipophilic to the tissues involve solubilizing the antioxidant in solvents such as benzyl alcohol, DMSO, or other chemicals. Not only does the presence of such solvents have the potential to introduce toxicities which may exacerbate microvascular injury, but their presence confuses the interpretation of any protocol designed to evaluate antioxidant effects.
The present invention seeks to overcome these limitations by solubilizing .alpha.-lipoic acid in aqueous solution without the use of additional solvents. .alpha.-lipoic acid and other antioxidants are rendered soluble in aqueous solutions by the use of ultrasonication. Because the .alpha.-lipoic molecule contains a polar (water soluble) carboxy-acid group and a non-polar, lipid soluble chain of carbon and sulfur atoms, the molecule is amphipathic, i.e., it has the ability to form micelles. Micelles may be formed in aqueous solution if a molecule possesses both polar and non-polar groups. After ultrasonication the polar, water soluble ends of the .alpha.-lipoic acid molecule are on the outside of aggregations of .alpha.-lipoic acid. The non-polar, lipid soluble tails are directed inward forming a tiny droplet, a micelle, which is water soluble. Ultrasonication of amphipathic molecules into micelles such as can be done with .alpha.-lipoic acid also has the possibility of creating mixed micelles. In this manner a mixture of .alpha.-lipoic acid with other antioxidants, which may not have the ability to form micelles alone for lack of any polar group, can be contained within a micelle of .alpha.-lipoic acid. In this way, mixed micelles containing .alpha.-lipoic acid and purely non-polar but highly lipid soluble antioxidants can be used to convey antioxidants to the tissues.
There are numerous other clinical conditions besides hemorrhagic shock which have as their final common pathway oxidant-inducing injury to tissues which can be treated and/or prevented with the inventive solutions. These conditions are described below.